P385: DETECTION OF KMT2A PARTIAL TANDEM DUPLICATIONS IN ACUTE MYELOID LEUKEMIA PATIENTS BY NGS
Authors: A. Artigas-Baleri, G. Oñate, A. Brell, J. Esteve, S. Vives, M. Tormo, M. Arnan, A. Garcia, R. Coll, A. Sampol, J. Bargay, F. Vall-llovera, O. Salamero, J. Nomdedéu, M. Pratcorona
Journal: HemaSphere (2022)

Background: Acute myeloid leukemia (AML) is a heterogeneous hematologic disease consisting of an aberrant proliferation and differentiation of hematopoietic stem cells in the bone marrow. In the last years the identification of cytogenetic and molecular biomarkers has improved the AML classification considering prognosis, and allowing a better risk-adapted treatment strategy. Regarding molecular biomarkers, NPM1, RUNX1 and ASXL1 mutations, FLT3-ITD (internal tandem duplications), CEBPAbi or KMT2A-PTD (partial tandem duplications) have been shown to have clinically significant prognostic value and they define specific AML disease subtype. Our study focuses on the detection of PTD in KMT2A which is involved in the regulation of gene expression during hematopoiesis. KMT2A-PTDs are found in 5-10% of normal karyotype patients and have been associated with poorer disease-free and overall survival rates. Mainly, PTDs in this gene span exons 2 to 8-10 and their detection is based on long-distance genomic PCR with extracted DNA from bone marrow. This is a time-consuming technique, and that is probably the main reason to explain why this marker is not widely performed at the time of diagnosis. Aims: We aimed to analyze the potentiality of Next Generation Sequencing (NGS) in the identification of KMT2A-PTD, a technique that is being implemented in many laboratories and could be a good strategy to detect this marker. Methods: This study included patients from CETLAM group (Grupo Cooperativo de Estudio y Tratamiento de las Leucemias Agudas y Mielodisplásicas) with a diagnosis of AML in 2021 (from January to October) and age≤70. The study of KMT2A-PTDs was carried out with DNA samples through two different techniques: PCR and NGS. DNA was extracted from bone marrow with the AllPrep DNA/RNA Mini Kit (Qiagen®). For the PCR amplification we used both custom 8-exon forward primer and 2-exon reverse primer. NGS was performed by hybridization capture technology (Nonacus®) using a custom panel which includes 43 genes. MiSeq (Illumina®) system was used for sequencing and Datagenomics for analysing the results. Hybridization capture allows the comparison of the number of reads per position between samples to identify structural variants as PTDs. This approach permits to know exactly which exons have been duplicated. Results: We included a total of 72 AML patients who were genotyped for KMT2A-PTD with both PCR and Hybridization capture NGS. We identified 4 and 7 patients with a partial tandem duplication in KMT2A gene by PCR and NGS, respectively. The four KMT2A-PTDs identified with PCR were also detected by NGS. In contrast, three PTDs were observed only with NGS. In two of them the PTD spanned exons 2 to 8 (Figure 1), while in the other it spanned exons 4 to 8. The first two PTDs were confirmed with the classic long-distance genomic PCR but performed with cDNA, skipping the presence of a large non-coding DNA in the rearrangement which could not be amplified by conventional PCR. To confirm the third PTD a new reverse primer in exon 4 was designed. All three NGS observed KMT2A-PTDs could be verified. Image:Summary/Conclusion: Our results show that Hybridization capture NGS is a good technique to detect KMT2A-PTDs, with a better sensibility than PCR. NGS is a cost-effective method as it is becoming an essential tool for AML classification at diagnosis.